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Single Lab Validation of a Method for the Determination of β‐
Galactooligosaccharides in Infant Formula & Adult Nutritionals / Sean Austin (NRC,
Lausanne)
25 Jul 2016
CONFIDENTIAL
∙ This document may not be reproduced or disclosed to third parties without prior authorization
Page 9 / 35
Enzyme‐treated Procedure
To the microtube containing 500 µL of sample solution add 25 µL of β‐galactosidase solution. Mix
well and place in a water bath at 60°C for 60 min. Cool sample to room temperature and continue
with 2AB Labelling.
2AB Labelling
Transfer 20 µL of sample to a 2‐mL microtube. Add 200 µL of 2AB labelling reagent and mix well.
Place the sample in a water bath at 65 °C for 2 hours. After incubation, mix well then cool sample at
4°C for 10 min.
Maltodextrin Hydrolysis
To each tube add 1 mL of ammonium acetate buffer and mix well. Transfer 500 µL of this solution to
a 2‐mL microtube and add 200 µL of AMG solution (60 U/mL). Mix well and incubate in a water bath
at 50°C for 30 min. After incubation, cool sample to room temperature and prepare sample for
chromatography.
Preparation for Chromatography
Add 700 µL of acetonitrile to the sample or standard and mix well. Centrifuge this solution at 10000
× g for 5 min, then transfer 1 mL of the supernatant to a vial suitable for the LC autosampler.
2.2.5
Preparation of Standard Curve
Prepare the solutions for the 6‐level standard curve by diluting the maltotriose stock solution (4
µmol/mL) as described in the following table:
Table 3: Dilution Scheme for the Preparation of the Standard Curve
Calibration Curve Dilutions in PHOSPHATE BUFFER (0.2 mol/L, pH 6.0)
Carbohydrate
# Std
Maltotriose
Stock volume
Final Volume
Standard
Concentration
[µL]
[mL]
[µmol/mL]
Maltotriose
Level #1
50
20
0.0100
Level #2
250
20
0.0500
Level #3
750
20
0.1500
Level #4
1120
10
0.4480
Level #5
1750
10
0.7000
Level #6
2250
10
0.9000
Preparation Procedure for Standards
:
For each standard, transfer 1000 µL of solution to a 2‐mL microtube, add 400 µL of laminaritriose
solution (0.3 µmol/mL) then take a 500 µL aliquot and follow the non‐treated procedure but stop
before the maltodextrin hydrolysis. Follow the maltodextrin hydrolysis procedure but add 200 µL of
ammonium acetate buffer (0.1 mol/L, pH 5.5) instead of AMG solution (i.e. no hydrolysis should
occur). Then continue with the preparation for chromatography.
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